Biological sensors are based on the immobilization of a recognition molecule at the surface of a transducer (a device that transforms the binding event between the target molecule and the recognition molecule into a measurable signal). In one prior approach, the transducer has been sensitive to any binding, specific or non-specific, that occurred at the transducer surface. Thus, for surface plasmon resonance or any other transduction that depended on a change in the index of refraction, such sensors have been sensitive to both specific and non-specific binding. Another prior approach has relied on a sandwich assay where, for example, the binding of an antigen by an antibody has been followed by the secondary binding of a fluorescently tagged antibody that is also in the solution along with the protein to be sensed. In this approach, any binding of the fluorescently tagged antibody will give rise to a change in the signal and, therefore, sandwich assay approaches have also been sensitive to specific as well as non-specific binding events. Thus, selectivity of many prior sensors has been a problem.
Another previous approach where signal transduction and amplification have been directly coupled to the recognition event is the gated ion channel sensor as described by Cornell et al., "A Biosensor That Uses Ion-Channel Switches", Nature, vol. 387, Jun. 5, 1997. In that approach an electrical signal was generated for measurement. Besides electrical signals, optical biosensors have been described in U.S. Pat. No. 5,194,393 by Hugl et al. and U.S. Pat. No. 5,711,915 by Siegmund et al. In the later patent, fluorescent dyes were used in the detection of molecules.
Despite the recent progress in such signal transduction and amplification directly coupled to a recognition event, further improvements have been desired especially in the development of optical biosensors.
One object of the present invention is an optical biosensor using a transduction approach that amplifies specific binding events thereby amplifying both sensitivity and specificity.
Another object of the present invention is to provide an optical biosensor and process for use of optically tagged receptor molecules to trigger signal transduction and amplification by a recognition event.
Still another object of the present invention is to provide an optical biosensor and process for signal transduction based on aggregation of receptor molecules through either binding by a multivalent protein or binding of multiple receptor molecules to the same protein or protein clusters.
Yet another object of the present invention is an optical biosensor and process using optical signal transduction resulting from an internal reference by virtue of a simultaneous increase in a red fluorescence and a decrease in a blue fluorescence. This can be coupled with an isobestic point that can be used to reference the absolute intensity of fluorescence.
Another object of the present invention is the fabrication of biomimetic membranes (supported and hybrid phospholipid bilayers) containing the functionalized (optically tagged) receptor molecules such as glycolipid receptor molecules.
Another object of the present invention is to functionalize naturally occurring glycolipid receptors (e.g., Gal.beta.1-3GalNAc.beta.1-4Gal(3-2.alpha.NeuAc).beta.1-4Glc.beta.1-1Cer, (GM1) with an optical tag such as fluorescent dye molecules.
Another object of the present invention is an optical biosensor and process using donor and acceptor dye molecules each of which is covalently attached to the recognition element to effect energy transfer upon aggregation following binding by a multivalent protein.
Another object of the present invention is an optical biosensor and process using hydrophobic optical tags (dye molecules) and hydrophobic linker molecules that can attach the optical tags to, e.g., a glycolipid so that the dye molecule stably resides in the upper leaf (layer) of a bilayer, e.g., a phospholipid bilayer. This can minimize non-specific interactions of the dye molecule with interferents.
Another object of the present invention is to provide an optical biosensor and process using optical transduction triggered by a specific protein binding event which results in energy transfer yielding both a decrease in the fluorescence of one dye species (e.g., blue emission) with a concomitant increase in the fluorescence of another dye species (e.g., red emission).